A variety of diseases are characterized partly by alteration in the pattern or amount of phosphate in regulatory or structural proteins. Protein phosphate content generally is controlled by phosphate addition, which is catalyzed by kinases, and by phosphate removal, which is catalyzed by phosphatases. Whereas kinases most often are regulated with great specificity, protein phosphatases are characteristically less selective. Thus, whereas kinases generally trigger systemic events in response to rather singular, specific stimuli, and then generally do so only through one signaling pathway, phosphatases exhibit broad dephosphorylating activity toward many of the phosphoproteins in their environment. Because of their specificity, kinases have always seemed especially attractive targets for drug development. For exactly the same reason, phosphatases, because of their lack of selectivity and broad systemic activities, have been viewed unfavorably as drug development targets.
Nevertheless, a number of attempts have been made to use phosphatase modulators for therapeutic purposes. For instance, Schieven in U.S. Pat. Nos. 5,565,491 and 5,693,627 reports on the use of phosphotyrosine phosphatase inhibitors to control proliferation of immune B cells. The patents disclose inhibitors that act directly on the phosphatase: metal-organic coordinate covalent compounds, nonhydrolyzable phosphotyrosine analogs, the streptomyces protein phosphatase inhibitor Dephostatin, and the prostatic acid phosphatase inhibitor 4-(fluoromethyl)phenyl phosphate. The inhibitors, as disclosed in the patents, inhibited proliferation of B cell leukemia and lymphoma cells, but also inhibited proliferation of normal B cells.
Lazo et. al. in U.S. Pat. Nos. 5,700,821, 5,856,506, and 5,925,660 discloses synthetic phosphatase inhibitors produced by combinatorial synthesis using L-glutamic acid as the initial scaffold. As disclosed in the patents, the compounds inhibited a variety of protein phosphatases, including PP1, PP2A, PP3, CDC25A, and CDC25B, and inhibited proliferation of human breast cancer cells in culture.
Hemmings discloses in U.S. Pat. No. 6,159,704 modulation of the phosphatase activity of the catalytic subunit of PP2A (“PP2Ac”) via its interaction with eRF1. As disclosed, eRF1 is the ribosome-associated factor responsible for polypeptide chain release at the termination of protein synthesis; but, it also binds to and interacts with the catalytic subunit of PP2A. According to the disclosure, eRF1 recruits PP2A to the ribosome and mediates the role of PP2A in protein synthesis. According to the patent, the inhibitors disrupt the interaction between eRF1 and PP2Ac. As further disclosed in the patent, they, thus, might inhibit protein synthesis, and therefore, might be useful to reduce aberrantly high protein synthesis and cell proliferation which, accordingly, might make them useful for treating proliferative disorders.
Yet another example in this regard is disclosed by Honkanen et. al. in U.S. Pat. No. 5,914,242. As disclosed in the patent, inhibitors of certain serine/threonine protein phosphatases, in particular fostriecin, an organic compound first isolated from streptomyces, is used to reduce damage to the heart following myocardial infarction. According to the patent, fostriecin inhibits PP2A thereby causing greater phosphorylation of the protein 1-2. This leads to proteolysis of 1-2 and reduces its level in the cell, because the phosphorylated protein is a much more active substrate for the protease. Since 1-2 inhibits PP1, the decrease in 1-2 activity due to proteolysis results in increased PP1 activity. According to the disclosure, increased PP1 activity protects cells from the deleterious effects of ischemia, although the mechanism of protection is not known. Further according to the disclosure, the protective effect of fostriecin might be due to inhibition of phosphatase activity that results in less dephosphorylation of proteins phosphorylated by protein kinase C.
The inhibitors in all of the foregoing patent disclosures, except Honkanen, act directly on the phosphatase, inhibiting its activity competitively or irreversibly. The inhibitor disclosed by Honkanen acts specifically to disrupt the interaction of the phosphatase, PP2Ac, with a ribosomal protein and likely will affect primarily the action of PP2Ac on protein synthesis, rather than its more general action as a phosphatase. In any case, all of the inhibitors of the foregoing patents act solely to decrease the activity of phosphatases. Inherently they cannot act to increase phosphatase activity, although, this is desirable in many cases.
Alzheimer's Disease (AD) is a progressive neurodegenerative disease associated clinically with memory impairment and decreased cognitive function [Selkoe, 2001 #2]. Post-mortem brains of AD patients display two pathological hallmarks: neuritic plaques and neurofibrillary tangles (NFTs). The plaques are extracellular deposits. They are composed of amyloid b-protein (Ab), which is a peptide derived from proteolytic cleavage of the amyloid precursor protein. NFTs, in contrast, are found primarily within the cell body. They are composed, in large part, of filaments of tau protein.
Tau normally is found predominantly in the axons of neurons where it stabilizes microtubules (MTs) and promotes their polymerization [Buee, 2000 #3]. MTs play a major role in maintaining the cellular architecture of neurons and are largely responsible for axonal transport [Goldstein, 2000 #4]. The integrity of MT structure is therefore critical for proper neuronal function and synaptic transmission. While tau normally is phosphorylated, it is abnormally hyperphosphorylated in NFTs [Grundke-lqbal, 1986 #47]. Increased phosphorylation appears to precede and promote NFT formation [Alonso, 2001 #5] [Alonso, 1996 #10]. Hyperphosphorylated tau is also found in the cytobsol of NFT-containing neurons [Kopke, 1993 #49]
Phosphorylation inhibits tau's ability to bind and stabilize MTs [Bramblett, 1993 #7] [Biernat, 1993 #8] [Alonso, 1994 #9]. Furthermore, hyperphosphorylated tau has a dominant negative effect in that it promotes MT disassembly by binding normal tau, MT associated protein 1, and MT associated protein 2, interfering with the ability of these three proteins to stabilize MTs [Alonso, 1997 #48]. These effects help account for the observation that neurons containing NFTs lack MTs. The cytoskeletal disruption brought about by hyperphosphorylated tau thus provides an explanation for its role in the neurodegeneration associated with AD.
Genetic evidence supports the conclusion that a critical event in the development of AD-type dementia is tau hyperphosphorylation [Lee, 2001 #6]. Though under some conditions Ab accumulation has been shown to promote NFT formation [Lewis, 2001 #11] [Gotz, 2001 #12], plaque formation is not essential for NFT-associated dementias, the so-called ‘tauopathies’. Mutations in the tau gene underlie several familial neurodegenerative diseases where filamentous deposits of hyperphosphorylated tau have been observed in the absence of amyloid plaques, most notably fronto-temporal dementia and Parkinsonism linked to chromosome 17 [Lee, 2001 #6].
Tau hyperphosphorylation results from an imbalance between kinase and phosphatase activities. Phosphorylation is catalyzed by the neuronally enriched serine/threonine kinases glycogen synthase kinase 3b (GSK-3b) and cyclin-dependent kinase 5 (CDK5) [Buee, 2000 #3] [Billingsley, 1997 #13]. The most important tau dephosphorylating enzyme is protein phosphatase 2A (PP2A) [Planel, 2001 #14] [Merrick, 1997 #15] [Kins, 2001 #16] [Gong, 1994 #50].
Recent results suggest that a decrease in PP2A activity, rather than increased kinase activities, is crucial for the elevated levels of tau phosphorylation associated with NFT formation. PP2A expression has been found to be significantly reduced in the hippocampus of AD brains relative to control brains [Vogelsberg-Ragaglia, 2001 #17], and expression studies in mouse brain indicate a general decrease in PP2A expression levels with age [Jiang, 2001 #18]. Treatment of cultured human neurons with the PP2A inhibitor okadaic acid results in tau hyperphosphorylation, reduced binding of tau to MTs, MT depolymerization, and axonal degeneration [Merrick, 1997 #15]. Moreover, starved mice display a pattern of tau hyperphosphorylation similar to that found in AD brains [Planel, 2001 #14], and this hyperphosphorylation appeared to result from decreased PP2A activity towards tau rather than an increased kinase activity. In fact, the tau phosphorylating activities of CDK5 and GSK-3b decreased under these conditions. Thus, reduced PP2A activity towards tau must be part of any model accounting for NFT formation during the progression of AD.
PP2A is a multimeric protein complex consisting of a 65 kDa A subunit that acts as a scaffold for the association of a 36-kDa catalytic C subunit and one of a variety of regulatory B subunits [Janssens, 2001 #19]. B subunits control the substrate specificity and subcellular localization of PP2A. Ba, the major regulatory subunit in brain [Kamibayashi, 1994 #20], targets trimeric PP2A to MTs [Sontag, 1995 #21] and dramatically increases the enzyme's activity towards the tau protein [Sontag, 1996 #22]. ABaC heterotrimers bind directly to the carboxyl-terminal MT binding domain of tau [Sontag, 1999 #23]. The highly conserved carboxyl-terminal sequence of the PP2A C subunit is a focal point for the enzyme's regulation. Reversible methyl esterification of the C-terminal leucine a-carboxyl group of the PP2A C subunit is a major locus of control [Tolstykh, 2000 #26] [Wu, 2000 #27] [Yu, 2001 #28] [Wei, 2001 #29].
PP2A methylation is controlled by a specific S-adenosylmethionine (SAM) dependent methyltransferase [Lee, 1993 #24] and a specific methylesterase [Lee, 1996 #25]. Methylation modulates PP2A activity by controlling the association of regulatory B subunits with the catalytic AC core [Tolstykh, 2000 #26] [Wu, 2000 #27] [Yu, 2001 #28] [Wei, 2001 #29]. The assembly of ABC heterotrimers proceeds as a multistep process with AC dimer methylation followed by binding of regulatory B subunits (FIG. 1). Tolstykh et al. [Tolstykh, 2000 #26] demonstrated that methylation of AC dimers from bovine brain dramatically increases their affinity for Ba regulatory subunits. Given the critical role of Ba in targeting PP2A activity towards tau, a decrease in PP2A methylation could lead to tau hyperphosphorylation, NFT formation, and neurodegeneration.
Decreased PP2A activity can contribute not only to tau hyperphosphorylation, but it can lead to other clinical indicators. For instance, homocysteine, through SAH hydrolase, is an end product of SAM-dependent methylation. The hydrolase reaction is reversible, and actually favors condensation of homocysteine and adenosine to form SAH (S-adenylhomocysteine). SAH is a potent inhibitor of methylation and accumulation of homocysteine (Hcy) thus generally is accompanied by increased SAH and, consequently, is associated with decreased methylation activity. Therefore, high plasma homocysteine levels generally may be indicative of decreased protein methylation and resultant decreases in methyl-dependent protein activities, such as PP2A phosphatase.
Indeed, over the last several years data has emerged in the clinical literature demonstrating a significant correlation between elevated plasma homocysteine (Hcy) and the occurrence of AD [Seshadri, 2002 #1] [McCaddon, 1998 #30] [Clarke, 1998 #31]. Elevated plasma Hcy has long been established as an independent, graded risk factor for cardiovascular disease [Clarke, 1991 #32] [Boushey, 1995 #33] [Welch, 1998 #34]; but, its role in AD has taken longer to establish. An early study found that patients with pathologically confirmed AD had significantly elevated plasma Hcy levels relative to a control group [Clarke, 1998 #31]. Hcy levels in the AD patients remained stable over time even as the disease progressed, suggesting that the elevation was not a result of neurodegeneration. Furthermore, patients with high plasma Hcy displayed more rapid neural atrophy over the course of three years than did patients with lower levels. More recent data from a prospective study provides convincing evidence that a rise in plasma Hcy precedes the onset of AD and is an independent risk factor for the disease [Seshadri, 2002 #1]. Baseline plasma Hcy levels were measured in 1092 non-demented patients and the occurrence of AD in this group was followed for several years. After adjusting for other AD risk factors, the authors found that plasma Hcy levels greater than 14 micromolar coincided with a roughly two-fold increased risk of developing AD. Further, elevated plasma Hcy appears to be a graded risk factor, with a 40 percent increased risk of developing AD associated with each 5 micromolar incremental rise. These studies clearly indicate a connection between high plasma Hcy and AD. While it has been recognized that insight into the mechanism underlying the association could give important clues for treatment of the disease, recognition of the association thus far has not provided a better understanding of AD or any other disease.
Elevated plasma homocysteine has been established as a risk factor not only for AD but also for heart disease, Type 2 diabetes, obesity, multiple sclerosis, stroke, cancer, rheumatoid arthritis, vascular disease, and birth defects; as well as various neurological illnesses including, among others, Parkinson's, depression, schizophrenia, and alcoholism. The relationship between elevated serum homocysteine and underlying disease etiology has not been elucidated for any of these diseases. Perhaps because of this, establishing the link has not led to effective therapeutic modalities, as yet. The general situation for all the diseases in this regard is fairly well illustrated by the foregoing discussion relating to Alzheimer's Disease. Presently nutritional supplementation is the only intervention thus far available for altering plasma homocysteine levels, and thereby, perhaps, reducing risk factors for these diseases. Unfortunately, whatever the efficacy of nutritional intervention at reducing plasma homocysteine, there is no evidence as yet that nutritional intervention actually reduces the homocysteine associated risk factor for disease. Furthermore, nutritionally forced reductions in plasma homocysteine actually may have deleterious effects.
Clearly, there is a need for an improved understanding of: the link between homocysteine levels, disease risk factors and disease itself, its etiology, and the factors that control the development and progress of the diseases. Even more important and pressing is the need for better diagnostic tools and for, above all, effective therapies for AD. Unfortunately, AD is merely illustrative in this regard. Many other diseases also are poorly understood, hard to diagnose, and presently lacking effective treatments.
Heretofore, proteins such as PP2A did not seem promising targets for effective therapeutics. Typically, they are ubiquitous, abundant and, perhaps worse for drug development, they are important general regulators of protein phosphokinase or protein phosphatase activities that affect virtually all phosphoproteins, and they interact with a very wide range of regulatory proteins. Thus, It appeared likely that targeting them would only lead to general systemic distress. Furthermore, it seemed likely that modulators of phosphatase activity would suffer some of the same disadvantages as the inhibitors discussed above. These inhibitors disadvantageously target a broad spectrum of serine/threonine proteases involved in regulating and performing vital cell processes and, consequently, broadly affect cellular metabolism and physiology, often with undesirable or deleterious consequences. Given their similar ubiquity and regulatory role, the same disadvantages were expected to limit the usefulness and efficacy of agents that modulate the activities of phosphatases. Thus, there has been and there continues to be a real need for improved diagnostics, better agents for altering the activities of proteins important in disease process, and effective methods for treating disorders and diseases such as AD.